Compositions and methods for capturing, isolating, detecting, analyzing and quantifying macromolecules

ABSTRACT

A method for making an imprint composition capable of capturing a macromolecule of interest, comprising the steps of (a) solidifying a fluidic matrix material capable of retaining shaped cavities when in a solid or semi-solid form in the presence of a template molecule that corresponds to a portion of the macromolecule, thereby yielding a solid or semi-solid matrix material having template molecules entrapped therein; and (b) removing the template molecules from the solid or semi-solid matrix material, yielding an imprint composition capable of capturing the macromolecule.

1. FIELD OF THE INVENTION

The present invention is directed to molecular imprints able toselectively bind macromolecules. The molecular imprints of the presentinvention can be prepared without obtaining a purified sample of atarget macromolecule. A template molecule possessing the structure of aportion of the macromolecule is synthesized and then imprinted.Molecular imprints made by this method form selective complexes withtheir target macromolecules. Arrays of molecular imprints can be used torapidly and inexpensively screen diverse biological samples.

2. BACKGROUND OF THE INVENTION

The recent explosion in the number of novel macromolecules, manyidentified by the genome sequencing efforts, has intensified the needfor improved compositions and methods for separating macromolecules.Thousands of recently identified macromolecules have yet to be purifiedand characterized functionally. Techniques for the rapid capture,isolation, detection, analysis, and quantification of macromoleculeswould accelerate the functional characterization of novelmacromolecules.

In particular, when the macromolecule is a protein, current methods ofcapture and separation are cumbersome and expensive. In one currenttechnique, affinity matrices are used to capture and/or separate aprotein of interest from a mixture of proteins and other molecules.Affinity matrices might be prepared using a purified sample of theprotein to create antibodies. However, the preparation of antibodiesthat specifically bind a protein can take several months and might evenrequire a purified sample of the protein. Alternatively, an affinitymatrix might be prepared using knowledge of the function of themacromolecule. For instance, an affinity matrix based on a bindingpartner of the protein might be used for capture and separation of theprotein. Unfortunately, methods of separating macromolecules thatrequire extensive knowledge about the macromolecule, or even a purifiedsample of the macromolecule, are ineffective with macromolecules thathave yet to be characterized. The requirement of a purified sample ofthe macromolecule for the preparation or selection of an affinity matrixoften presents researchers with nothing more than frustratingcircularity.

In addition to affinity matrices, other techniques are also used toseparate macromolecules. For example, the current state of the arttechnique for separating large numbers of proteins is two-dimensionalgel electrophoresis. The technique typically resolves about 2,000proteins, and the best gels can resolve up to 11,000 proteins (Abbot,1999, Nature 402:715-720). Unfortunately, many researchers requireseparation techniques that can resolve proteins from samples as diverseas the entire protein fraction of a mammalian cell. The protein fractionof a cell can contain tens of thousands of proteins, overwhelming theresolving power of 2D electrophoresis (Abbot, 1999, supra). Since thefull sequences of the genomes of many species, including humans, arenearing completion, researchers must now grapple with the functions ofhundreds of thousands of novel macromolecules (Abbot, 1999, supra). Newtechniques of macromolecular separation that require limited informationor even no information about the target macromolecules are needed.

Currently, researchers are seeking improvements in protein separation.For instance, some are attempting to create chips which specificallybind proteins. In one typical chip, antibodies specific for knownproteins are attached to a substrate to form a microarray. These chipscan then be used to bind and identify proteins from a complex solution(Abbot, 1999, supra). However, these chips suffer from the samelimitations of antibody production that plague affinity matrices. Foreach protein to be bound by the chip, a unique antibody must be preparedby an expensive process that can take several months. In addition, manyproteins are not sufficiently immunogenic to create antibodies forbinding.

In the field of small molecules, the technique of molecular imprintinghas provided an efficient method for the preparation of matrices thatare capable of selectively binding a target molecule. To prepare amolecular imprint, a matrix is formed around a template molecule. Afterthe matrix has formed and the template molecule is removed, the matrixcan then be used to selectively bind the template molecule. As early as1949, a silica gel was created that selectively bound a dye (Dickey,1949, Proc. Natl. Acad. Sci. USA 35:227-229). Recently, an imprintprepared with phenyl-α-D-mannopyranoside was sufficiently selective toresolve a racemic mixture of the saccharide (Wulff, 1998, supra).

Current methods form imprints of molecules in organic polymers (Wulff,1998, Chemtech 28:19-26). To create cavities of defined shape,polymerizable molecules are bound, covalently or noncovalently, to atemplate molecule (Wulff, 1998, supra). The resulting complex iscopolymerized in the presence of a large amount of a cross-linkingreagent (Wulff, 1998, supra). The templates are then removed leavingmicrocavities with defined shapes and arrangements of functional groups(Wulff, 1998, supra). Imprints made by such a technique displayselective binding for the template molecule. Molecular imprints havebeen used for chromatographic separation, immunoassays, chemosensors,and even catalysis (Wulff, 1998, supra).

To date, molecular imprints have had limited application to the bindingof larger molecules including macromolecules. In fact, one review statesthat only small molecules can be imprinted with any great confidence.Molecular imprints of larger molecules like nucleic acids, peptides,proteins and cells fail because larger molecules yield moreheterogeneous binding sites and because larger molecules can be toofragile for conventional methods of molecular imprinting (Cormack andMosbach, 1999, Reactive and Functional Polymers 41:115-124).

Nevertheless, a few successful imprints of larger molecules have beenproduced. Synthetic polymers which selectively bind amino acidderivatives and peptides were created using the target amino acidderivative or peptide as a template (Kemp, 1996, Anal. Chem.68:1948-1953). Imprints have also been created which bind to nucleotidederivatives (Spivak and Shea, 1998, Macromolecules 31:2160-2165). Ionicmolecular images of polypeptides have been created by mixing a matrixmaterial with the intact polypeptide chain to be bound by the molecularimage (U.S. Pat. No. 5,756,717). Molecular imprints of cytochrome c,hemoglobin and myoglobin, respectively, have been prepared bypolymerizing acrylamide in the presence of each intact protein (U.S.Pat. No. 5,814,223). An imprint of horse myoglobin selectively boundhorse myoglobin from a mixture of proteins including whale myoglobin(U.S. Pat. No. 5,814,223).

Although current methods of molecular imprinting have shown limitedinitial success at selectively binding macromolecules, the currentmethods are not sufficient for the efficient capture of macromolecules.The current techniques for molecular imprinting require a purifiedsample of the macromolecule to be bound by the imprint. The inability toproduce a specific imprint in the absence of a purified sample of themacromolecule is no different from one of the failings of conventionalmethods of protein separation. In addition, current methods of preparingmolecular imprinting are not amenable to creating the thousands ofimprints often required by current large-scale experiments. Purificationof hundreds or thousands of proteins to create a matrix for separatingthe proteins of a cell extract is no more efficient that 2Delectrophoresis. An efficient method for producing compositions thatselectively bind macromolecules given limited information about thestructure or function of those macromolecules is needed. An ideal methodcould produce a composition capable of binding a macromolecule given aslittle information as a partial primary structure of the macromolecule.

An improvement in molecular imprinting to enable the preparation ofaffinity matrices in the absence of a purified sample of themacromolecule would overcome many limitations of the art of molecularimprinting. Techniques are also needed to efficiently separate andidentify thousands of proteins. Compositions with specificity formacromolecules that can be produced rapidly and at low cost will enablesuch techniques. Ideal compositions would be arrays of such bindingcompositions, each composition designed to bind a given macromolecule.Such an array could be used to rapidly screen a complex biologicalsample for a number of different macromolecules simultaneously.

3. SUMMARY OF THE INVENTION

These and other shortcomings in the art are overcome by the instantinvention, which in one aspect provides imprint compositions useful forcapturing, isolating, detecting and/or quantifying macromolecules in asample. Generally, the imprint compositions comprise a matrix materialdefining an imprint of a template molecule. The template moleculetypically corresponds to a portion of a macromolecule of interest, suchas, for example, a polynucleotide, a polypeptide, polysaccharide, aprotein, a glycoprotein, a receptor, an enzyme, a nucleic acid, acarbohydrate, etc. If the macromolecule is composed of n identifiablestructural units as defined below, then the template molecule cancorrespond to a portion of the macromolecule that includes from 1 up to(n−1) of those structural units. Alternatively, the template moleculecan correspond to as little as 1% of the macromolecule or to as much as99% of the macromolecule. The portion to which the template moleculecorresponds may be an internal portion of the macromolecule or aterminal portion of the macromolecule. Alternatively, the portion may bea side-group or modification of the macromolecule, such as apolysaccharide group of a glycoprotein macromolecule, or a portionthereof. Preferably, the template molecule will correspond to acontiguous terminal portion of the macromolecule.

Matrix materials that can comprise the imprint compositions of theinvention include substances that are capable of undergoing a physicalchange from a fluid state to a semi-solid or solid state. In the fluidstate, the particles of a matrix material move easily among themselves,and the material retains little or no definite form. A matrix materialin the fluid state can be mixed with other compounds, including templatemolecules. In the semi-solid or solid state, the matrix materials arecapable of forming and retaining cavities that complement the shape oftemplate molecules. Examples of such matrix materials include heatsensitive hydrogels such as agarose, polymers such as acrylamide, andcross-linked polymers.

The imprint compositions of the invention may take a variety ofdifferent forms. For example, they may be in the form of individualbeads, disks, ellipses, or other regular or irregular shapes(collectively referred to as “beads”), or in the form of sheets. Eachbead or sheet may comprise imprint cavities of a single templatemolecule, or they may comprise imprint cavities of two or more differenttemplate molecules. In one embodiment, the imprint composition comprisesimprint cavities of a plurality of different template molecules arrangedin an array or other pattern such that the relative positions of theimprint cavities within the array or pattern correlate with theiridentities, i.e. the identities of the template molecules used to createthem. Each position or address within the array may comprise an imprintcavity of a single template molecule, or imprint cavities of a pluralityof different template molecules, depending upon the application.Moreover, the entire array or pattern may comprise unique imprintcavities, or may include redundancies, depending upon the application.

As discussed above, the template molecule used to make the imprint willtypically correspond to a portion of a macromolecule of interest.However, as will be discussed more thoroughly below, an important aspectof the invention includes the ability to use the imprint compositions ofthe invention to isolate novel macromolecules from complex mixturesand/or samples. In this embodiment, a template molecule can have astructure that does not necessarily correspond to a portion of any knownmacromolecule. Rather, the template molecule could have a structure thatcorresponds to a portion of a consensus sequence derived from a familyof macromolecules. Alternatively, the template molecule might have arandom structure. A molecular imprint of a template molecule can bind anovel macromolecule if the template molecule corresponds to a portion ofthe novel macromolecule. An array of imprints of template molecules canbe used to rapidly screen a mixture for novel macromolecules. An arrayof imprints of the complete set of polymeric template molecules composedof a defined number of monomers can be used to capture most or all ofthe macromolecules of a mixture.

In another aspect, the present invention provides methods of making theimprint compositions of the invention. According to the method, acompound or mixture of compounds that is capable of undergoing a changeof physical state such that the resultant product is a solid orsemi-solid matrix capable of retaining shaped cavities is contacted witha template molecule under conditions in which the change of physicalstate is effected. Changing the physical state of the compound ormixture of compounds in the presence of the template molecule results ina solid or semisolid matrix having the template molecules entrappedtherein. The template molecules are then removed, yielding a solid orsemisolid matrix defining cavities that correspond in shape to thetemplate molecules. This resultant product is a molecular imprintcomposition. Particularly preferred methods of making molecular imprintcompositions include the method of surface imprinting described incopending application Ser. No. 09/507,299, filed concurrently herewith,which is incorporated herein by reference.

In still another aspect, the present invention provides methods of usingthe imprint compositions to capture, isolate, detect, analyze and/orquantify a macromolecule of interest in a sample. According to themethod, a sample suspected of containing a macromolecule of interest iscontacted with an imprint composition of the invention under conditionsin which the macromolecule binds the imprint composition. Theimprint-macromolecule complex may be optionally rinsed to remove unboundcomponents of the sample. The macromolecule may be dissociated from thecomplex and isolated and/or quantified. Alternatively, the presence ofthe macromolecule may be detected, and/or its quantity determined,without dissociating it from the complex.

The methods can be used to capture macromolecules of known, partiallyknown or unknown structure. In the former two embodiments, the imprintcomposition comprises an imprint of a template molecule that correspondsto a known portion of the macromolecule of interest. In the latterembodiment, the imprint may comprise an imprint of a template moleculethat corresponds to a conserved portion of a specific class ofmacromolecules, such as for example, a conserved portion of a receptorsuperfamily or family, or it may comprise a predicted sequence or acompletely random sequence. These latter imprint compositions can beused to capture and/or isolate novel members of known classes ofmacromolecules, or completely new types of macromolecules.

Macromolecules may be detected, captured, isolated, analyzed and/orquantified according to the methods of the invention singly, using animprint composition specific for a particular macromolecule of interest,or alternatively, pluralities of different macromolecules can becaptured simultaneously from a complex mixture using, for example, thearray or pattern imprint compositions described herein, for subsequentdetection, isolation and/or quantification.

The methods and compositions of the invention provide significantadvantages over currently available protein separations and molecularimprinting technologies. Unlike known imprinting techniques, themolecular imprints of the present invention do not require a purifiedsample of a target macromolecule for preparation. The primary structureof a portion of the macromolecule is sufficient to create an imprintthat can specifically capture the macromolecule. If the targetmacromolecule is novel, a molecular imprint of template molecules thatdo not necessarily correspond to portions of known macromolecules can beused to screen for the target macromolecule. Because they do not requireisolation of the macromolecule of interest, the molecular imprints ofthe present invention can be prepared in far less time and at a fractionof the cost of conventional protein separation media.

The methods and compositions of the invention also have widespreadapplicability, ranging from the detection and/or isolation of specificmacromolecules of interest from samples, to the capture, isolation,analysis and/or quantification of pluralities of macromolecules fromcomplex mixtures for applications such as, for example, expressionprofiling, to the discovery of novel members of known classes ofmacromolecules and/or completely new types of molecules altogether.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A illustrates a macromolecular target that can be captured with animprint composition of the present invention;

FIG. 1B illustrates a polymeric macromolecular target;

FIG. 1C illustrates a polymeric macromolecular target with asuperstructure;

FIG. 2 illustrates the preparation of a molecular imprint of a templatemolecule that corresponds in structure to a portion of a macromoleculeand subsequent capture and isolation of the macromolecule;

FIG. 3A illustrates a one-dimensional surface imprint array;

FIG. 3B illustrates a two-dimensional array of surface imprint beadsdistributed on a substrate;

FIG. 3C illustrates a cross-sectional view of an embodiment of a surfaceimprint array;

FIG. 4 illustrates the capture of a plurality of macromolecules with oneembodiment of a molecular imprint array;

FIG. 5 provides an SDS-PAGE analysis of a capture experiment performedwith a molecular imprint according to the present invention;

FIG. 6 provides an SDS-PAGE analysis of a capture and isolationexperiment performed with a surface imprint of the present invention;and

FIG. 7 provides an SDS-PAGE analysis of experiments capturing andisolating cytochrome c from a mixture and from a cell lysate withsurface imprints of the resent invention.

5. DETAILED DESCRIPTION OF THE INVENTION

Current methods of macromolecular separation are inefficient whenapplied to novel macromolecules. Small molecules have been successfullycaptured and separated with molecular imprints for many years (Wulff,1998, supra). However, molecular imprinting has limited application tothe separation of macromolecules. Only a few imprints of macromoleculeshave been described, and those imprints required purified samples of themacromolecule for preparation. There is a need for improvement in thefield of macromolecular separation, and the field of molecularimprinting requires improvement to be useful for the separation ofmacromolecules.

The present invention provides compositions and methods that overcomethe limitations of macromolecule separation and molecular imprinting.The invention is based, in part, on the Applicant's discovery that amolecular imprint made with a template molecule that corresponds instructure to a portion of a macromolecule of interest can be used tospecifically capture and/or isolate that macromolecule from a sample.Until Applicant's discovery, conventional techniques required a purifiedsample of the macromolecule to prepare a molecular imprint withspecificity for the macromolecule.

The present invention overcomes the limitations that prevented broadapplication of molecular imprinting to the capture of macromolecules.The use of molecular imprints is no longer limited to the subset ofmacromolecules for which a purified sample is available. Molecularimprinting can now be applied to capture macromolecules using only alimited amount of information about the macromolecule. If the primarystructure of as little as a portion of the macromolecule is available,then one can use the techniques of the present invention to rapidlycreate an inexpensive affinity medium with specificity for themacromolecule. For instance, when one has obtained the primary structureof a portion of a protein from the sequence of a nucleic acid encodingthe protein, one has enough information to create a molecular imprint tospecifically capture the protein.

5.1 Abbreviations

As used herein, the abbreviations for the genetically encodedL-enantiomeric amino acids are conventional and are as follows:

One-Letter Common Amino Acid Symbol Abbreviation Alanine A Ala ArginineR Arg Asparagine N Asn Aspartic acid D Asp Cysteine C Cys Glutamine QGln Glutamic acid E Glu Glycine G Gly Histidine H His Isoleucine I IleLeucine L Leu Lysine K Lys Methionine M Met Phenylalanine F Phe ProlineP Pro Serine S Ser Threonine T Thr Tryptophan W Trp Tyrosine Y TyrValine V Val

The abbreviations used for the D-enantiomers of the genetically encodedamino acids are lower-case equivalents of the one-letter symbols. Forexample, “R” designates L-arginine and “r” designates D-arginine. When apolypeptide sequence is represented as a series of three-letter orone-letter amino acid abbreviations, it will be understood that theleft-hand direction is the amino terminal direction and the right-handdirection is the carboxy terminal direction, in accordance with standardusage and convention.

5.2 The Invention

In one aspect, the invention provides imprint compositions comprising amatrix material having a cavity of a template molecule imprintedtherein/thereon. The template molecule typically corresponds to aportion of a macromolecule of interest, but may also have a structurethat corresponds to a consensus sequence from a family of macromoleculesor a random structure. The imprint composition can be used to detect thepresence of the macromolecule in a sample, to capture, isolate, detect,analyze and/or quantify the macromolecule from a sample.

5.2.1 Macromolecules

Macromolecules that can be captured, isolated, detected, analyzed and/orquantified using the imprint compositions of the invention include anytype of macromolecule from which a template molecule can be designed andconstructed according to the principles taught herein. Virtually anytype of macromolecule can be captured, isolated, detected, analyzedand/or quantified using the methods and compositions of the invention.Non-limiting examples include biological polymers such as polypeptides,polynucleotides and polysaccharides, non-biological polymers such aspolyesters, polyethers, polyurethanes, block co-polymers, and otherpolymers known to those of skill in the art and biological andnon-biological non-polymeric compounds such as antibiotics, steroids,natural products, dyes, etc. Thus, non-limiting examples of the myriadtypes of macromolecules that may be captured, isolated, detected,analyzed and/or quantified using the methods and compositions of theinvention include cytokines, hormones, growth factors, enzymes,cofactors, ligands, receptors, antibodies, carbohydrates, steroids,therapeutics, antibiotics, and even larger structures such as viruses orcells, and other macromolecular targets that will be apparent to thoseof skill in the art.

It will be understood that as used herein, the expression“macromolecule” is not intended to place specific size limitations uponthe molecules that may be captured with the imprint compositions of thepresent invention. Rather, “macromolecules” as used herein refers tomolecules that comprise a plurality of structural moieties such that atemplate molecule corresponding to at least one of the structuralmoieties can be used to prepare a molecular imprint capable of bindingthe macromolecule. Template molecules and the formation of a molecularimprints are discussed in more detail below.

The identities of the structural moieties that comprise macromoleculesas used herein will depend upon the nature of the macromolecule, and mayinclude regions of primary, secondary and/or tertiary structure of themacromolecule. For example, for polypeptide macromolecules thestructural moieties may be the individual amino acids composing thepolypeptide, or alternatively, if the polypeptide has several structuraldomains, as is often the case with, e.g., enzymes and antibodies, thestructural moieties may be the individual structural domains. Forexample, an antibody macromolecule may be viewed as being composed ofindividual amino acids, or Fab and Fc structural domains, oralternately, Fab′ and Fc structural domains, etc., depending upon theproteolytic enzymes used to digest the antibody. A glycosylatedpolypeptide may be viewed as being composed of individual amino acids orstructural domains as described above and/or saccharide oroligosaccharide structural moieties. A polynucleotide macromolecule maybe viewed as being composed of individual nucleotide structuralmoieties.

Structural moieties may also be regions of secondary structure, such asregions of α-helix, β-sheet, β-barrel, etc. of proteins or regions ofA-form helix, B-form helix, Z-form helix, triple helix, etc. ofpolynucleotides.

For non-polymeric macromolecules such as, for example, antibiotics, thestructural moieties may be the various core groups composing theantibiotic. For example, polyene antibiotics such as amphotericin B andnystatin may be viewed as being composed of polyene macrocycle andsaccharide structural moieties.

As will be discussed in more detail below, the macromolecule willpreferably comprise a combination of one or more structural moietiesthat, when taken together, uniquely identify the macromolecule overclosely related macromolecules. Template molecules derived from suchmacromolecules can be used to make imprint compositions according to theinvention that are capable of selectively capturing or binding themacromolecule from a complex mixture and/or sample.

Due to their ability to be uniquely defined by their primary structure(i.e., primary sequence of monomers), polymers are preferred templatemolecules according to the invention. The most preferred polymers arebiological polymers such as polypeptides, polynucleotides andpolysaccharides.

Polypeptides include polymers of two or more amino acids known to thoseof skill in the art, and derivatives known to those of skill in the artsuch as glycopeptides. Polynucleotides include polymers of two or morenucleotides and can be DNA or RNA of natural or synthetic origin.Polynucleotides can be single-stranded, double-stranded, or multiplystranded. Polysaccharides include polymers of two or more sugars and canbe linear or branched. The monomer subunits of biological polymers canbe those that occur naturally, or synthetic analogs of such monomersknown to those of skill in the art.

The macromolecules according to the invention may be derived fromvirtually any source. They may be obtained from natural sources such asbiological samples or from synthetic sources. In particular, the imprintcompositions of the present invention can be used to capture and isolatemacromolecules from biological sources as complex as a cell or tissuelysate. The imprint compositions can also be used to purify a syntheticmacromolecule from a mixture of precursors and byproducts. The imprintcompositions can even be used to resolve a racemic mixture of syntheticmacromolecules.

5.2.2 Template Molecules

The imprint compositions of the invention are prepared from a templatemolecule. In many embodiments, the template molecule has a structurethat corresponds to a portion of the macromolecule of interest. Atemplate molecule “corresponds” to a portion of the macromolecule if itpossesses the structural features of that portion of the macromoleculeand substantially no other structural features of the macromoleculeoutside that portion. The template molecule can possess structuralfeatures of the macromolecule by way of structural identity with theportion of the macromolecule. Alternatively, the template molecule canpossess structural features of the portion of the macromolecule byapproximating or mimicking the structure of at least one structuralmoiety of the portion of the macromolecule.

Template molecules that correspond to portions of known macromoleculescan be prepared according to known principles. In general, for any givenmacromolecule, a portion of the structure of the macromolecule is usedto prepare or select a template molecule that corresponds in structureto that portion of the macromolecule. For example, referring to FIG. 1A,a molecular imprint composition with specificity for macromolecule 2 canbe prepared with a template molecule that corresponds to structural unit4 of the macromolecule. For a polymeric macromolecule 6, as illustratedin FIG. 1B, a template molecule can correspond to contiguous sequence ofmonomers 7. Polymeric macromolecule 8, illustrated in FIG. 1C, has amacromolecular superstructure, and the template molecule can correspondto domain 9 of the macromolecule.

The correspondence between the topography of the template molecule andthe topography of the portion of the macromolecule should be closeenough so that the portion of the macromolecule fits specifically withinan imprint or a cavity formed by the template molecule (see, e.g., FIG.2). In general, for any macromolecule that comprises a plurality ofstructural groups, the template molecule can correspond to at least oneof the structural groups of the macromolecule. For example, if themacromolecule is an antibiotic such as erythromycin, a template moleculecan correspond to one of the sugar moieties of erythromycin. If themacromolecule is a polymer, a preferred template molecule would consistof a series of monomer units that is identical to a contiguous series ofmonomer units of a portion of the polymer.

For example, referring to FIG. 1B, when the macromolecule is apolypeptide, the template molecule may be a peptide having an amino acidsequence that identically corresponds to the amino acid sequence of acontiguous region of the polypeptide macromolecule. If the polypeptidemacromolecule is glycosylated, the template molecule may be anoligosaccharide having a primary sequence of saccharides thatcorresponds identically to the primary saccharide sequence of all or aportion of the glycosyl groups. If the macromolecule is apolynucleotide, the template molecule may be an oligonucleotide having anucleotide sequence that corresponds identically to the nucleotidesequence of a contiguous region of the polynucleotide macromolecule.When the polynucleotide is single-stranded, the template molecule willbe single-stranded. When the polynucleotide is double-stranded, thetemplate molecule can be either single-stranded or double-stranded,depending upon whether the resultant imprint composition will be used tocapture, isolate detect, analyze and/or quantify the polynucleotideunder native or denaturing conditions. If single-stranded, the templatemolecule may correspond to either strand of the double-strandedpolynucleotide macromolecule.

Those of skill in the art will recognize that a template molecule neednot have exact structural identity with the portion of the macromoleculein order to correspond to that portion. Often, a template molecule mayincorporate topographic substitutions. A substitution is “topographic”if the topography of the template molecule creates a cavity that bindsor captures the corresponding portion of the macromolecule. Preferably,a template including a topographic substitution creates an imprint thatspecifically binds the corresponding portion of the targetmacromolecule. Template molecules comprising topographic substitutions,and that therefore do not correspond identically to a portion of themacromolecule, are said to correspond substantially to themacromolecule. Thus, unless specifically indicated otherwise, as usedherein, the expression “corresponds to” is intended to encompass thosesituations where a template molecule corresponds identically orsubstantially to a macromolecule of interest.

When constructing a template molecule that does correspondssubstantially to a portion of the macromolecule, the template moleculeshould be topographically of a size that is similar to or larger thanthe portion of the macromolecule, so that the macromolecule can fitwithin or bind the imprint cavity created by the template molecule. Forexample, since Phe and Tyr have side chains of similar structure, andthe Phe side chain can be viewed as a “sub-set” of the Tyr side chain, atemplate molecule having a Phe or Tyr corresponds to a macromoleculePhe. Similarly, a template molecule Cys, Ser or Thr corresponds to amacromolecule Ser. Thus, Tyr is a topographic substitution of Phe, andSer and Thr are topographic substitutions of Cys. For the twentygenetically encoded amino acids, preferred corresponding template aminoacids are as follows:

TABLE OF CORRESPONDENCE Macromolecule Template Aliphatic Ala Ala, Val,Leu, Ile Val Val Leu Leu Ile Ile Non Polar Gly Gly, Ala Pro Pro Cys Cys,Ser, Thr Met Met, Lys, Arg Aromatic His His, Trp Phe Phe, Tyr Tyr TyrTrp Trp Polar Asn Asn, Gln Gln Gln Ser Ser, Cys, Thr Thr Thr Charged LysLys Arg Arg Asp Asp, Glu Glu Glu

Non-encoded amino acids and/or amino acid analogues that correspond toportions of a polypeptide macromolecule will be apparent to those ofskill in the art. In addition, for other types of macromolecules, thoseof skill in the art will recognize that template molecules can beselected or prepared with topographic substitutions according to theprinciples discussed above for polypeptide macromolecules. For example,an oligonucleotide macromolecule adenine can be topographicallysubstituted with 7-diazadenine; a macromolecule guanine with7-diazaguanine; a macromolecule cytosine with 5-methylcytosine, etc.Specific typographic substitutions will depend upon the specificmacromolecule and will be apparent to those of skill in the art.

The closeness of the correspondence between the template molecule andthe macromolecule of interest will depend upon the desired degree ofspecificity between the imprint and the target macromolecule. Templatemolecules that correspond identically to a portion of a macromoleculeare expected to exhibit the highest degree of specificity for themacromolecule. Thus, the closeness of correspondence will depend upon,among other factors, the particular application and the complexity ofthe sample, and will be apparent to those of skill in the art.Preferably, the template molecule will correspond identically to aportion of the macromolecule to be captured.

It has been discovered that the presence of reactive groups such assulfhydryl groups in template molecules can be disadvantageous for thepreparation of molecular imprints. Nevertheless, the imprintcompositions of the present invention can be prepared to capturemacromolecules that contain such reactive groups. The imprintcompositions of the present invention can be prepared so efficiently andinexpensively that a number of techniques can be applied to suchmacromolecules. To avoid including such reactive groups, a template canbe designed that corresponds to a portion of the macromolecule that doesnot contain the reactive group. However, if a portion of themacromolecule is selected that includes a reactive group, the templatemolecule can be designed to include a topographic substitution for thereactive group. In particular, macromolecular Cys residues can besubstituted to Ser residues in the template molecule. Alternatively,reagents can be used to reduce or block the reactive groups of themacromolecule and of the corresponding template molecule. Such reagentsare known to those of skill in the art. For example, any template Cysresidue can be reduced with dithiothreitol or β-mercaptoethanol orblocked with reagents that prevent the formation of inter molecular orintramolecular disulfide bridges such as N-ethylmaleimide, iodoaceticacid or other reagents known to those of skill in the art. When thetemplate molecules are “blocked” in this fashion, the reactive groups inthe macromolecule to be captured are preferably blocked with the samereagent, as a higher degree of specificity during capture will beachieved. For example, to capture a polypeptide macromolecule whichincludes disulfide bridges with a molecular imprint prepared with atemplate molecule in which the Cys residues are blocked, the disulfidebridges of the macromolecule should be reduced prior to, or concomitantwith, contacting the macromolecule with the molecular imprint.Preferably, the sulfhydryl groups of the reduced Cys residues will befurther blocked with the same reagent used to block the template Cysresidues.

Those of skill in the art will also recognize that in many instancescompounds that mimic the structures of other compounds are known. Thetemplate molecule may comprise, in whole, or in part, such mimeticstructures. Mimetic compounds that can be used to create templatemolecules, as well as their use to create template molecules, will beapparent to those of skill in the art.

For example, peptidomimetic compounds that mimic the structures ofpeptides are well known. One class of peptidomimetics includes the classof compounds in which the amide linkage of the peptide chain arereplaced with isosteres of amides. Isosteres of amide bonds generallyinclude, but are not limited to, —CH₂NH—, —CH₂S—, —CH₂CH₂—, —CH═CH—(cisand trans), —C(O)CH₂—, —CH(OH)CH₂—and —CH₂SO—. Compounds having suchnon-amide linkages and methods for preparing such compounds arewell-known in the art (see, e.g., Spatola, March 1983, Vega Data Vol. 1,Issue 3; Spatola, 1983, “Peptide Backbone Modifications” In: Chemistryand Biochemistry of Amino Acids Peptides and Proteins, Weinstein, ed.,Marcel Dekker, New York, p. 267 (general review); Morley, 1980, TrendsPharm. Sci. 1:463-468; Hudson et al., 1979, Int. J. Prot. Res.14:177-185 (—CH₂NH—, —CH₂CH₂—); Spatola et al., 1986, Life Sci.38:1243-1249 (—CH₂—S); Hann, 1982, J. Chem. Soc. Perkin Trans. I.1:307-314 (—CH═CH—, cis and trans); Almquist et al., 1980, J. Med. Chem.23:1392-1398 (—COCH₂—); Jennings-White et al., Tetrahedron. Lett.23:2533 (—COCH₂—); European Patent Application EP 45665 (1982) CA97:39405 (—CH(OH)CH₂—); Holladay et al., 1983, Tetrahedron Lett.24:4401-4404 (—C(OH)CH₂—); and Hruby, 1982, Life Sci. 31:189-199(—CH₂—S—). The template peptides may include one or more of such amideisosteres or combinations of such isosteres.

Additionally, one or more amide linkages can be replaced withpeptidomimetic or amide mimetic moieties which do not significantlyinterfere with the structure or activity of the peptides. Suitable amidemimetic moieties are described, for example, in Olson et al., 1993, J.Med. Chem. 36:3039-3049. All that is required is that the threedimensional surface of the mimetic template compound have a threedimensional surface with sufficient correspondence to the surface of themimicked portion of the macromolecule to create a cavity thatspecifically fits the portion of the macromolecule.

The template molecule may correspond to any portion of themacromolecule, including an internal region, a terminal region, or amodifying molecule such as a polysaccharide of a glycoprotein.Preferably, the portion of the structure of the macromolecule is ofsufficient size that the macromolecule forms a tight complex with themolecular imprint. Also preferably, the portion of the macromolecule issufficiently unique that the molecular imprint is selective for themacromolecule. For instance, if the macromolecule is to be captured froma complex mixture, a preferred template molecule corresponds to aportion of the macromolecule that uniquely defines that macromoleculeover other macromolecules in the mixture. Such a unique portion of themacromolecule can be determined by comparing the structure of themacromolecule with the structures of known macromolecules of the complexor by statistical analysis. When the macromolecule is a polypeptide, atemplate molecule consisting of a sequence of seven amino acids canprovide a molecular imprint that is highly selective for themacromolecule.

In general, template molecules can correspond to any portion of themacromolecule, as long as the template does not correspond to the entiremacromolecule. If the macromolecule consists of n identifiablestructural units, then the portion of the macromolecule to which thetemplate corresponds can consist of from 1 up to (n−1) of thosestructural units. Preferably, the structural units of the macromoleculeto which the template molecule corresponds are contiguous within theprimary structure of the macromolecule. If one of skill in the art canidentify a terminus or termini in the primary structure of themacromolecule, then a preferred template molecule corresponds to atemplate that includes a terminus of the macromolecule. Alternatively,the portion of the macromolecule to which the template moleculecorresponds can be expressed in size as a fraction of the size of theentire macromolecule. For example, template molecules can correspond toa portion of the macromolecule that consists of from 1% to 5%, from 1 to10%, from 1 to 15%, from 1 to 20%, from 1 to 25%, from 1 to 30%, from 1to 35%, from 1 to 40%, from 1 to 50to 60%, from 1 to 70%, from 1 to 80%,from 1 to 90%, from 1 to 95%, or from 1 to 99% of the structure of theentire macromolecule. Preferably, template molecules have a primarystructure that corresponds to a contiguous portion of the primarystructure of the macromolecule. If the macromolecule is a polymer, thestructure of the template molecule can correspond to a sequence ofmonomers from the polymer.

In instances where the macromolecule is a polymer, the portion of thepolymer to which the template molecule corresponds can also be expressedas a range of monomer units of the polymer. If the polymer consists of nmonomer units, then the portion of the polymer, to which the templatemolecule corresponds, can consist of up to (n−1) monomer units.Alternatively, the portion of the polymer can consist of from 1 to 50monomer units, from 2 to 40 monomer units, from 3 to 30 monomer units,from 3 to 15 monomer units, from 3 to 10 monomer units, from 4 to 10monomer units, from 4 to 9 monomer units, from 4 to 8 monomer units,from 4 to 7 monomer units, or from 5 to 7 monomer units. If the polymeris branched, then the portion can be branched or unbranched. Preferably,the portion of the polymer is a contiguous sequence of monomers from theprimary structure of the polymer.

If the macromolecule is a polypeptide, the template molecule cancorrespond to a portion of the polypeptide that consists of a sequenceof amino acids selected from the primary sequence of the polypeptide.For instance, if the polypeptide has a length of n amino acids, theportion of the macromolecule to which the template molecule correspondscan consist of up to (n−1) amino acids of the primary structure of thepolypeptide. Alternatively, the portion of the polypeptide can consistof a range of amino acids from the primary structure of the polypeptideconsisting of from 1 to 50 amino acids, from 2 to 40 amino acids, from 3to 30 amino acids, from 3 to 15 amino acids, from 3 to 10 amino acids,from 4 to 10 amino acids, from 4 to 9 amino acids, from 4 to 8 aminoacids, from 4 to 7 amino acids, or from 5 to 7 amino acids. Preferredportions of the macromolecule are those that consist of a continuoussequence of amino acids from the primary structure of the polypeptide.When the macromolecule is a polypeptide, the preferred template moleculecorresponds to the contiguous sequence of seven amino acids at thecarboxy terminus of the polypeptide. Template molecules that correspondto the amino-termini are less preferable because polypeptides frombiological sources often have heterogeneous modifications at theiramino-termini.

When the macromolecule is a polypeptide modified with a polysaccharide,the template molecule can be a polysaccharide having a sequence ofsaccharides selected from the primary sequence of the polysaccharide. Ifa contiguous polysaccharide component of the polypeptide contains nsaccharide units, then the selected sequence of saccharides can containup to n saccharides. Alternatively, the selected sequence can containfrom 1 to 50 saccharides, 2 to 40 saccharides, 3 to 30 saccharides, 3 to15 saccharides, 3 to 10 saccharides, 4 to 10 saccharides, 4 to 9saccharides, 4 to 8 saccharides, 4 to 7 saccharides, or 5 to 7saccharides. If a polysaccharide component of the polypeptide isbranched, the template molecule can also be branched. A preferredtemplate molecule corresponds to a contiguous sequence of saccharideunits from the polysaccharide, whether branched or unbranched. Thetemplate molecule can also correspond to a hybrid structure selectedfrom the primary structure of polypeptide consisting of at least oneamino acid and at least one saccharide.

When the macromolecule is a polynucleotide, the template molecule can bean oligonucleotide having a sequence of nucleotides selected from theprimary sequence of the polynucleotide. If the polynucleotide has nnucleotides, then the selected sequence of nucleotides can have a lengthfrom 1 to (n−1) nucleotides. Alternatively, the selected sequence cancontain from 1 to 50 nucleotides, 2 to 40 nucleotides, 3 to 30nucleotides, 3 to 15 nucleotides, 3 to 10 nucleotides, 4 to 10nucleotides, 4 to 9 nucleotides, 4 to 8 nucleotides, 4 to 7 nucleotides,or 5 to 7 nucleotides. Preferably, the selected sequence is a contiguoussequence of nucleotides from the primary sequence of the polynucleotide.

When the macromolecule is any type of polymer, the selected sequence ofmonomers for the structure of the template molecule can be chosen froman internal region of the polymer or from a terminus of the polymer. Ifthe polymer has unique termini, the sequence of monomers can also bechosen from any terminus of the polymer. If the macromolecule ispolypeptide, the preferred template molecule for the present inventioncorresponds to the sequence of amino acids at the carboxy terminus ofthe polypeptide.

For macromolecules that are non-polymeric such as, for example,antibiotics, the template molecule preferably corresponds to astructural feature that uniquely identifies the antibiotic. For example,if several antibiotics differ in structure from one another by theidentity of a single substituent (e.g., a sugar residue, a lipid moiety,etc.), then a molecular imprint prepared with a template molecule thatcorresponds to that unique feature can be used to specifically capturethat antibiotic from a complex mixture of related antibiotics. Forexample, the antibiotic amphotericin B (AmB) can be selectively capturedfrom a mixture of AmB and amino sugar derivatives thereof with amolecular imprint prepared with a template that corresponds to the aminosugar moiety of amphotericin.

If mixtures of several distinct macromolecules are to be captured, thetemplate can correspond to a common structural feature. For example, amixture of AmB and amino sugar derivatives thereof can be captured witha molecular imprint prepared with a template molecule that correspondsto their common polyene core.

Due to their ease of manufacture and their inexpensiveness, suchmolecular imprints can be used for industrial scale isolation andpurification of macromolecules, and have broad-ranging applicabilitytoward the separation of complex mixtures of natural products whosecomponent molecular species have similar physiochemical properties.

5.2.3 Template Molecules Useful for Preparing Imprints that can CaptureNovel Macromolecules

In those embodiments above where template molecules correspond toportions of known macromolecules, the molecular imprints of the presentinvention are most useful for capturing known macromolecules. However,in another important embodiment, the present invention is also usefulfor capturing, isolating, detecting, and quantifying novelmacromolecules. In this embodiment, template molecules that do notnecessarily correspond to a portion of a known macromolecule can be usedto capture novel macromolecules, even those for which no structuralinformation is known.

A novel macromolecule is a macromolecule for which limited or nostructural or functional information is available. If any structuralinformation is available, a molecular imprint can be prepared using atemplate molecule that corresponds to the portion of the availablestructural information as described above. The template molecule canalso correspond to all of the available structural information. When nostructural information is known about a macromolecule, but it is knownto be functionally related to a known macromolecule, the templatemolecule can correspond to a portion of a macromolecule having similarfunction, the template molecule can correspond to a portion of amacromolecule with similar function, or the template molecule cancorrespond to a consensus sequence of a family of macromolecules withsimilar function. In addition, for any novel macromolecule, even one forwhich no structural or functional information is available, a molecularimprint of a template molecule with a random structure might be able tocapture the novel macromolecule. For example, an as yet unidentifiedmacromolecule can be captured, isolated, detected, analyzed, quantifiedand/or identified from a complex sample with such a molecular imprint.

In the present embodiment of the invention, a template molecule can be apeptide, a polynucleotide, a branched or unbranched polysaccharide, or amimic or derivative of such molecules. When template molecules withrandom structures are used, a set of template molecules with randomstructures is particularly useful. For instance, the complete set ofrandom peptides of a defined length can be used to generate a completeset of molecular imprints complementing the peptide template molecules.This complete set of molecular imprints can be used to screen samplesfor novel polypeptides.

The size of a template molecule should be appropriate for creating animprint cavity that can specifically fit a portion of a novelmacromolecule. In general, a novel macromolecule can be captured with amolecular imprint of a template molecule with a mass of 100 Da to 5000Da. If a certain class of macromolecule is targeted, then the structureof that class of macromolecule can be used to design or select thestructure of template molecules useful for generating the molecularimprints to capture novel macromolecules of the class. For instance, ifthe novel macromolecule target could be a polymer, then a templatemolecule can consist of from 3 to 30 monomer units of the polymer. Ifthe novel macromolecule target could be a polypeptide, then a templatemolecule can consist of from 3 to 30 amino acids. If the novelmacromolecular target could be a polysaccharide, then the templatemolecule can consist of from 3 to 30 saccharides. If the novelmacromolecule target could be a glycosylated polypeptide, than atemplate molecule can consist of from 3 to 30 amino acids, from 3 to 30saccharides, or a hybrid structure with from 3 to 30 amino acids orsaccharides. If the target macromolecule could be a polynucleotide, thenthe template molecule can consist of from 3 to 30 nucleotides.

Template molecules of the invention may be synthesized or obtained byvirtually any means. For example, the template molecule may be obtainedfrom commercial sources, synthesized using standard solution orsolid-phase synthetic schemes and/or isolated from biological samples.For example, oligosaccharide, oligonucleotide and peptide templatemolecules may be obtained commercially or synthesized according tostandard techniques. Template molecules that correspond to completefunctional domains of, e.g., polypeptide or polynucleotidemacromolecules may be obtained by enzymatic cleavage. For example, Fabtemplate molecules may be obtained by enzymatic cleavage of antibodymacromolecules. Double-stranded oligonucleotide template molecules maybe obtained by endonucleolytic cleavage of polynucleotidemacromolecules. The actual technique used to obtain the templatemolecule will depend upon the identity of the template molecule, andwill be apparent to those of skill in the art.

5.2.4 Formation of an Imprint

The imprint compositions of the invention can be prepared according toany of the known techniques for making molecular imprints, with oneimportant modification. Instead of creating an imprint with themacromolecule to be captured, the imprint compositions of the inventionare created with a template molecule. Non-limiting examples of suitabletechniques that can be used in conjunction with the invention aredescribed, e.g., in U.S. Pat. Nos. 5,858,296; 5,786,428; 5,587,273;5,821,311; 5,814,223; and 5,757,717; 5,994,110; 5,959,050; 5,916,445;5,872,198; 5,814,223; 5,728,296; 5,630,978; and 5,310,648, thedisclosures of which are incorporated herein by reference.

A general method for preparing an imprint composition of the presentinvention is illustrated in FIG. 2. Referring to FIG. 2, a templatemolecule 12 is contacted with a matrix material 14 under conditions inwhich template molecule 12 becomes entrapped or embedded within matrixmaterial 14, yielding complex 16. As illustrated, the template molecule12 corresponds in structure to an internal portion of a macromolecule10. matrix material 14 is a compound or mixture of compounds that iscapable of undergoing a 35 change of physical state from a fluid form toa solid or semisolid form. Matrix material 14 may comprise virtually anycompound or mixture compounds that is compatible with template molecule12 and that is capable of undergoing a change of physical state to forma solid or semisolid such that the changed form is capable of retainingshaped cavities. The physical state change can be induced by virtuallyany means, including by thermal, chemical and/or electromagneticprocesses. Examples of suitable compounds are discussed below.

Preferably, the conditions under which the template molecule isimprinted are similar or identical to the conditions under which themacromolecule is to be captured. For instance, if the macromolecule isto be captured under denaturing conditions, then the template moleculeshould be imprinted under the same denaturing conditions. Similarly, ifthe macromolecule is to be captured under native conditions, then thetemplate molecule should be imprinted under the same native conditions.Native and denaturing conditions are well-known to those of skill in theart. Particular native and denaturing conditions for capturing certainmacromolecules are discussed in detail below.

During the embedding process, matrix material 14 changes physical statefrom a fluid state to a solid or a semisolid state 14′ in the presenceof template molecule 12. Fluid states are known to those of skill in theart and include those states where the molecules of matrix material 14move freely among themselves and where compound 14 retains little or nodefinite form. In the fluid state, template molecule 12 can mix freelywith the molecules of matrix material 14. In the solid or semisolidstate, matrix 14′ is sufficiently shape-retaining to retain cavitiesthat complement the shape of template molecule 12. Removal of templatemolecule 12 from complex 16, by, for example, extensive washing, yieldsimprint composition 18. In imprint composition 18, solid or semisolidmatrix 14′ defines cavities 12′ that complement the topography oftemplate molecule 12. Complex 16 and imprint composition 18 may comprisetemplate molecules 12 that are wholly embedded within matrix 14′ and aretherefore not removed during the removal step.

Although not illustrated, matrix material 14 can also be contacted witha plurality of different template molecules, each like template molecule12. Each template molecule can correspond to a portion of a differentmacromolecule yielding a variation of matrix 14′ that can capture aplurality of different macromolecules. Alternatively, each templatemolecule can correspond to a different portion of the same macromoleculeyielding a variation of matrix 14′ that can bind or capture themacromolecule at a plurality of positions.

In one embodiment, matrix material 14 may be a solid or semisolidcompound that liquifies upon application of heat and resolidifies whenthe heat is removed.

Referring to FIG. 2, to make an imprint composition according to theinvention using such a heat sensitive compound, template molecule 12 andheat sensitive compound 14 are mixed under conditions in which heatsensitive compound 14 liquifies. The heat source is then removed and, asthe liquid cools, it solidifies to form complex 16. Removal of templatemolecules 12 via, for example, diffusion, yields imprint composition 18.Of course, in order to maintain the integrity of cavities 12′, imprintcomposition 18 should be kept at temperatures below the liquificationtemperature of heat sensitive compound 14 during storage and subsequentmanipulations.

Many heat-sensitive compounds that can be used to make imprintcompositions according to the invention are known in the art andinclude, by way of example and not limitation, hydrogels such asagarose, gelatins, moldable plastics, etc.

Examples of other suitable hydrogels are described in U.S. Pat. Nos.6,018,033, 5,277,915, 4,024,073, and 4,452,892, the disclosures of whichare incorporated herein by reference. Preferably, the temperature atwhich the heat-sensitive compound 14 liquifies will be in a range thatdoes not destroy or otherwise substantially degrade the templatemolecule 12.

In another embodiment, matrix material 14 may comprise a compound ormixture of compounds that undergoes a chemical or light inducedliquid-to-solid state change. Examples of these types of compounds thatare suitable for use with the present invention include, but are notlimited to styrene, methyl methacrylate, 2-hydroxyethyl methacrylate,2-hydroxyethyl acrylate, methyl acrylate, acrylamide, vinyl ether, vinylacetate, divinylbenzene, ethylene glycol dimethacrylate, ethylene glycoldiacrylate, pentaerythritol dimethacrylate, pentaerythritol diacrylate,N,N′-methylenebisacrylamide, N,N′-ethylenebisacrylamide,N,N′-(1,2-dihydroxyethylene)bis-acrylamide, trimethylolpropanetrimethacrylate, vinyl cyclodextrin, and polymerizable cyclodextrin.Further examples of polymerizable compounds that are useful for thepreparation of molecular imprints can be found in U.S. Patent No.5,858,296, which is hereby incorporated by reference in its entirety.The preferred polymerizable substance for the present invention isacrylamide.

Preferably, matrix material 14 comprises a monomer or mixture ofmonomers that undergo chemical or light-induced polymerization to yielda solid or semisolid polymer matrix (“polymerizable compound”).Referring to FIG. 2, in order to prepare imprint compositions accordingto the invention with such polymerizable compounds 14, the templatemolecule 12 and a polymerizable compound 14 are mixed in a solvent thatis suitable for polymerization of polymerizable compound 14. Ifnecessary, an initiator for the polymerization of the polymerizablecompound is included. Optionally, the template molecule 12 can becovalently bound to the polymerizable compound 14, or the two can beallowed to form non-covalent complexes. Polymerization can by started byadding an appropriate catalyst such as ultraviolet radiation or freeradical initiation. After polymerization is complete, the templatemolecule 12 is removed by diffusion, incubation in a chaotropic reagentsuch as urea or guanidine, or by other techniques known to those ofskill in the art.

Cross-linking reagents can optionally be used with a polymerizablecompound 14 to confer rigidity to the molecular imprint. The presentinvention contemplates any ratio of polymerizable compound tocross-linking reagent that yields a molecular imprint of sufficientintegrity to form a cavity whose shape corresponds to the shape of thetemplate molecule. Cross-linking reagents are known to those of skill inthe art. Examples of such cross-linking reagents can be found in U.S.Pat. No. 5,858,296. Preferred molecular imprints are prepared withacrylamide and the cross-linking reagent ethylene glycol dimethacrylate(EGDMA) or with acrylamide and bisacrylamide.

In general, matrix material 14 and template molecule 12 can be contactedunder any conditions which permit matrix material 14 change physicalstate to a solid or semisolid matrix 14′. For instance, matrix material14 and template molecule 12 can be contacted under native conditions orunder denaturing conditions. “Native conditions” and “denaturingconditions” can be defined with respect to the template molecule or withrespect to the template molecule or with respect to the macromoleculeaccording to principles known to those of skill in the art. Preferably,the conditions of contacting matrix material 14 and template molecule 12are similar or identical to the conditions of contacting macromolecule10 and matrix 14′.

The concentration of matrix material 14, template molecule 12, and anoptional cross-linking reagent are not critical for success and can bedetermined according to principles known to those of skill in the art ofmolecular imprinting. The previously cites references provide guidancefor choosing appropriate concentrations for specific matrix materials.The number of cavities 12′ in matrix 14′ can be adjusted by varying theconcentration of template molecule 12. Generally, the concentration oftemplate molecule 12 can vary widely, ranging from as low as 0.01 mM toas high as 1 M. Although the concentration of the template molecule isnot critical, template molecule concentrations of about 1 mM produceeffective molecular imprints.

Once the matrix is in a solid or semi-solid state, the molecularimprints can be processed to take on a variety of shapes. Usually, themolecular imprint will initially take on the same shape as the containerused to create matrix 14′. However, any shape that might be useful forcapturing macromolecules is possible. For example, they may be in theform of individual beads, disks, ellipses, or other regular or irregularshapes (collectively referred to as “beads”), or in the form of sheets.Beads can be formed by grinding a rigid matrix 14′ or by suspension anddispersion techniques. Methods of making imprinted beads are discussedin Damen et al., 1980, J. Am. Chem. Soc. 102:3265-3267; Braun et al.,1984, Chemiker-Zeitung 108:255-257; and Bystrom et al., 1993, J. Am.Chem. Soc. 115:2081-2083. Imprinted beads may also be prepared byimprinting in the pore network of preformed beaded silica as discussedin Wulff et al., 1985, Reactive Polymers 3:261-2757. Dispersiontechniques are discussed in Sellergren et al., 1994, 673:133-141. Theformation of beaded molecular imprints by suspension polymerization isdescribed in U.S. Patent No. 5,821,311. All of these references areincorporated herein by reference.

5.2.5 Formation of a Surface Imprint

In a preferred embodiment, the molecular imprints of the presentinvention are surface imprints. Surface imprint compositions aremolecular imprint compositions in which a substantial number or fractionof the imprint cavities are localized at or near the surface of theimprint. Surface cavities are more accessible to macromolecules and areoriented to facilitate binding. Because of the high density of imprintcavities, surface imprints have greater capacity for bindingmacromolecules.

A detailed description of surface imprints and methods for theirpreparation are described in copending Application Ser. No. 09/507,299,supra, which is hereby incorporated by reference in its entirety.Briefly, surface imprints may be prepared by forming the imprintcavities around an immobilized template molecule. Alternatively, surfaceimprints can be prepared by a two-phase method. In the two-phase method,a conjugate molecule is prepared that can partition to the interface oftwo phases. The conjugate molecule comprises a template moiety thatconstitutes a template molecule as described above and a tail moiety.The template moiety is soluble in one phase of the two phase system, anda matrix material 14 is soluble in the same phase. The tail moiety issoluble in the other phase of the two phase system. When the matrixmaterial changes physical state, a matrix 14′ is formed definingoriented surface cavities that complement the shape of the templatemoiety. These surface imprints can be used in any the methods of thepresent invention.

5.2.6 Arrays of Imprints

The present invention also provides arrays of imprints and/or imprintcompositions. The arrays may be comprised of a plurality of individualimprint compositions arranged in an array or pattern, or may comprise asingle piece or sheet of matrix material having a plurality of imprintcavities imprinted thereon. In this latter embodiment, the imprintcavities are arranged in an array or pattern. The arrays may beone-dimensional, two-dimensional or three-dimensional. For instance, ifthe array comprises individual beads, a one-dimensional array can beprepared by introducing the beads into a capillary tube. Atwo-dimensional array can be prepared by distributing the beads into thewells of a microtiter plate and/or by affixing the individual beads ontoa substrate.

For example, referring to FIG. 3A, the array can be an ordered patternof individual beads 50 where each bead 50 is an imprint composition ofthe invention. As illustrated in FIG. 3A, the individual beads 50 may bedisposed within a housing 52. Housing 52 can serve the dual purpose ofretaining the ordering of individual beads 50 and providing a structurethrough which the sample may be flowed. The ends of the capillary tubemay be optionally plugged with, for example, glass wool, a frit, orother porous materials to hold the beads in the tube during sample flow.

Alternatively, individual beads 50 could be distributed, either singlyor in pluralities, amongst the wells of, for example, a microtiterplate, or affixed to the surface of a substrate, such as a glass plate,plastic sheet or film, etc. For example, referring to FIG. 3B,individual imprints 50 (in this case illustrated as square pads) can beaffixed onto a glass sheet 54 in an ordered two-dimensional matrix.Methods for fixing polyacrylamide pads that can be adapted to createarrays according to the invention are described in U.S. Pat. No.5,552,270. Methods of affixing other types of matrix materials ontosubstrates are well-known and will be apparent to those of skill in theart.

As illustrated by the above examples, a key feature of the arrays of theinvention is the ability to correlate the identity of a particularimprint with its relative position within the array. The identity of animprint is defined by the identity of the template molecule used tocreate the imprint. Thus, in the array illustrated in FIG. 3B, theidentity of a particular imprint 50 is identifiable by itsxy-coordinates within the array. In the array illustrated in FIG. 3A,the identity of a particular imprint 50 is identifiable by itsx-coordinate within the array. This feature is particularly useful fordetecting, capturing, analyzing, isolating and/or quantifyingpluralities of macromolecules from complex samples, as will be discussedin more detail, below.

The arrays of the invention also include matrices which have pluralitiesof imprint cavities disposed at defined relative positions. For example,referring to FIG. 3C, a single sheet of matrix material 14′ may comprisean ordered arrangement of imprint cavities 12′.

In the arrays of the invention, each array element or address (i.e.,each set of array coordinates) may be unique, i.e., each address in thearray may contain an imprint of a different template molecule, oralternatively, the array may comprise redundancies. Moreover, while inmany instances each array element will comprise imprint cavities of asingle template molecule, one or more of the array elements may compriseimprint cavities of 2 or more different template molecules.

The number of elements comprising the array can vary over a wide range,from as few as 2 to as many as 10, 10², 10³, 10⁴, 10⁵, 10⁶ or even more,and is limited only by the ability to make an array having the desiredcomplexity, as will be described in more detail, below.

The spatially identifiable arrays of the invention provide the abilityto screen and/or analyze complex samples. For example, referring to FIG.4, imprint array 40 is constructed from template array 30. Templatearray 30 comprises a plurality of template molecules 31, 33, 35 and 37which uniquely correspond to macromolecules 32, 34, 36 and 38,respectively. Imprint array 40 comprises matrix 14′ which definescavities 31′, 33′, 35′ and 37′ which correspond to template molecules31, 33, 35 and 37, respectively. Because template array 30 is spatiallyidentifiable (i.e., the identities of the template molecules are knownor identifiable by their coordinates or relative positions within thearray), imprint array 40 is also spatially identifiable. Moreover, sincethe template molecules uniquely correspond to their respectivemacromolecules, imprint array 40 can be used to detect the presence ofmacromolecules 32, 34, 36 and 38 in a sample. For example, referring toFIG. 4, imprint array 40 is contacted with a sample comprisingmacromolecules 32, 34 and 39 under conditions in which themacromolecules bind, or become captured by, their respective imprintcavities. Macromolecules 32 and 34 are captured at locationscorresponding to cavities 31′ and 33′. When the array is scanned forbound macromolecules, the presence of macromolecules at the addressescorresponding to templates 31 and 33 reveals that the sample containedmacromolecules 32 and 34. Macromolecule 39 is not detected, as imprintarray 40 does not contain an address or element capable of binding thismacromolecule. The relative amounts of macromolecules 32 and 34 in thesample can be optionally determined by quantifying the amount ofcaptured macromolecules at each address, as will be described in moredetail below.

A spatially identifiable array of molecular imprints is particularlyuseful when an array of molecular imprints of template molecules that donot necessarily correspond to portions of known macromolecules is usedto screen a complex mixture in order to isolate novel macromolecules.Partial structural information about any captured novel macromoleculecan be deduced from the position at which it binds the array. In orderfor capture to occur, a portion of the captured novel macromolecule musthave a structure that corresponds to the structure of the templatemolecule that was used to create the imprint at that position in thearray. If the captured, novel macromolecule is a protein and themolecular imprint is an imprint of a peptide template molecule, then aportion of the amino acid sequence of the captured macromolecule mighteven be identical to the amino acid sequence of the peptide templatemolecule.

5.2.7 Methods of Making Arrays

The arrays of the invention can be readily prepared using standardtechniques available in the art. Arrays comprised of individual imprintbeads may be prepared by any method of dispensing and/or affixing thebeads in a spatially defined fashion known in the art. The imprint beadsare prepared according to the previously described methods. Arrayscomprised of a single piece or sheet of matrix are typically preparedusing an array of immobilized template molecules. The array of templatemolecules can be prepared according to any of the techniques well-knownto those of skill in the art. For example, an array of immobilizedtemplate molecules may be prepared by synthesizing each templatemolecule on an individual synthesis substrate such as a glass bead orother solid-phase synthesis resin and affixing the individual synthesissubstrates to another substrate such as a glass or plastic sheet or filmin an ordered array or pattern without cleaving the template moleculesfrom the synthesis substrates. Such substrates of immobilized templatemolecules may be prepared individually, or pluralities of differentimmobilized template molecules may be prepared simultaneously using oneof the numerous combinational synthesis strategies known in the art(see, e.g., U.S. Patent Nos. 6,001,579; 5,968,736; 5,961,923; 5,925,562;5,789,172; Fodor et al., 1991, Science 251:767-773; Brenner and Lerner,1992, Proc. Natl. Acad. Sci. USA 81:5381-5383; Amoto, 1992, Science257:330-331; Lam et al., 1991, Nature 354:82-84; Houghton et al., 1991,Nature 354:84-86; Jung and Beck-Sickinger, 1992, Angew. Chem. Int. Ed.Engl. 91:367-383; and Kerr et al., 1993, J. Amer. Chem. Soc.,115:2529-31).

Methods for making myriad different types of immobilized templatemolecules are well-known. For example, methods for synthesizing peptidetemplate molecules immobilized on synthesis substrates are described inMerrifield, 1997, Meth. Enzymol. 289:3-13; methods for synthesizingoligonucleotide template molecules immobilized on synthesis substratesare described in Southern et al., 1994, Nuc. Acids Res. 22:1368-1373. Aplethora of reactions available for synthesizing a wide variety of othertypes of immobilized template molecules are described in Bunin, 1998,The Combinational Index, Academic Press, San Diego, Calif.Alternatively, the arrays of the invention may be prepared from an arrayof template molecules in which each template molecule is immobilized tothe same substrate.

Such template arrays can be prepared according to well-known techniques.For example, the template array may be prepared by spotting templatemolecules onto a substrate under conditions in which the templatemolecule covalently or non-covalently attached to the substrate usingany of the spotting devices described in U.S. Pat. Nos. 5,601,980,6,001,309, 5,785,926, and 4,877,745. Any of these devices, or otherdevices useful for dispensing small aliquots of liquids into substrates,can be adapted for use to create the desired array of templatemolecules.

Alternatively, the array of template molecules may be prepared bysynthesizing in situ each template molecule at its desired address orlocation within the array. Several in situ synthesis methods useful formaking arrays of template molecules have been described in the art. Forinstance, an array of peptides immobilized on a substrate can beprepared according to, for example, U.S. Pat. Nos. 5,958,703; 5,919,523;5,847,105; or 5,744,305. An array of oligonucleotides immobilized on asubstrate can be prepared according to, for example, U.S. Pat. Nos.5,919,523; 5,843,655, 5,143,854; 5,847,105; 5,837,832; 5,770,722; PCTapplication No. WO 92/10092; or PCT application No. WO 90/15070.

A significant advantage of preparing the arrays of the invention fromtemplate arrays is the dimensions that can be achieved. Template arraysprepared by spotting or in situ synthesis methods can readily beprepared that have synthesis spots or features on the order of 10-100μm, permitting the synthesis of tens of thousands, or even millions, ofdifferent template molecules in a substrate area measuring about 1 cm oneach edge (see, e.g., Fodor et. al., 1991, supra). Imprint arrayscreated with such template arrays will have similar dimension andcomplexities. Thus, imprint arrays capable of capturing tens ofthousands, hundreds of thousands or even millions of uniquemacromolecules that measure only 1 cm² can be readily prepared. Theability to create such miniature array imprints makes it possible, forthe first time, to analyze the plethora of macromolecules present incomplex samples such as cells. Due to their miniature dimensions, verylittle sample is required for analysis. Moreover, since template arraysof different types of template molecules can be prepared (e.g., an arraycomprising both peptide and oligonucleotide template molecules),different types of macromolecules can be captured and analyzedsimultaneously.

In instances where the array is prepared with an array of immobilizedtemplate molecules, the template molecules can be optionally attached tothe support via a labile linkage. The array of molecular imprints canthen be prepared by forming imprints, according to one of the methodsdescribed above, in the presence of the array of immobilized templatemolecules. The template molecules can be cleaved from the support priorto removing the support from the newly formed molecular imprints.Cleavable linkages can be cleaved with chemicals, enzymes, orelectromagnetic radiation. If the linkage can be cleaved withelectromagnetic radiation and the transition of matrix material can alsobe induced by electromagnetic radiation, the wavelength of the radiationthat cleaves the linkage should be compatible with the wavelength of theradiation that induces the transition of the matrix material. Cleaving alabile linker allows the template molecules to be removed from themolecular imprints, according to one of the methods described above,with minimal disruption of the integrity of the molecular imprints. Theremaining portions of the template molecules can be removed by diffusionor by incubation in a chaotropic reagent such as urea or guanidine or byother techniques known to those of skill in the art for disruptingmolecular complexes. Cleavable linkers and/or linkages suitable forattaching template molecules are known to those of skill in the art.Appropriate examples are described, for instance, in U.S. Pat. Nos.5,766,556; 5,095,084; 6,013,440; 5,962,337; and 5,958.703 thedisclosures of which are hereby incorporated by reference.

5.2.8 Methods of Capturing Macromolecules

Also within the scope of the present invention are methods of usingmolecular imprints to capture macromolecules. Molecular imprints usefulfor capturing macromolecules can are prepared as described above. Tocapture a macromolecule, the macromolecule or a mixture comprising themacromolecule is contacted with the molecular imprint under conditionsin which the macromolecule binds the imprint. A macromolecule “binds” acavity if it becomes entrapped or immobilized within the cavity in aspecific manner such that it is specifically captured from a compositioncomprising it. Preferably, the molecular imprint is an imprint of atemplate molecule that corresponds to a portion of the macromolecule.For capture, the imprint compositions may be disposed within a housingto create a chromatography column, or used batch-wise. Alternatively,the imprint is an imprint of a template molecule that does notnecessarily correspond to a portion of a known macromolecule. An imprintof such a template molecule is useful for capturing a macromoleculewhose structure has yet to be determined.

Also preferably, the conditions for contacting the macromolecule withthe imprint are similar to or identical to the conditions under whichthe imprint was formed. While not intending to be bound by anyparticular theory, it is believed that the molecular imprints of thepresent invention capture macromolecules because the portion of themacromolecule that corresponds to the template is captured by the cavityof the imprint that formed around the template molecule. If the captureconditions are similar to the imprinting conditions, the portion of themacromolecule is more likely to adopt a structure similar to thestructure of the corresponding template molecule.

The choice of conditions depends on the macromolecule and the templatemolecule that corresponds to a portion of the macromolecule. When themacromolecule is a protein and the template molecule corresponds tosequence of amino acids of the protein, the preferred capture conditionsare often denaturing. However, when a template molecule corresponds to alarge fragment of a protein, such as a pepsin fragment of animmunoglobulin, then native imprinting and capture conditions will oftenyield superior results. When the macromolecule is a double-strandedpolynucleotide, the preferred capture conditions are native conditionsthat allow the macromolecule to maintain its native structure. When themacromolecule is a single-stranded polynucleotide, the captureconditions may be native or denaturing conditions. One of skill in theart will recognize whether native or denaturing conditions areappropriate. In situations where the choice of imprinting and captureconditions is not clear, the molecular imprint compositions of thepresent invention can be prepared so efficiently and inexpensively thata series of conditions can be assayed to determine the ideal conditions.

The exact conditions to retain a native or denatured structure arewell-known and will be apparent to those of skill in the art. Forinstance, denaturing conditions for polypeptides can include SDS, urea,guanidine, or any other protein denaturant known to those of skill inthe art. Denaturing conditions for polynucleotides can include hightemperature, formamide, high ionic strength, and other conditions knownto those of skill in the art.

A plurality of macromolecules can be captured simultaneously bycontacting the macromolecules with an array of the invention. The amountof a macromolecule in a sample can be quantified by capturing themacromolecule with a molecular imprint and determining the amount of themacromolecule captured by the imprint. Techniques for detecting acaptured macromolecule or quantifying the amount of a capturedmacromolecule include infrared spectroscopy, UV spectroscopy, visiblespectroscopy, surface acoustic wave devices, refractive index,evanescent wave sensors, bulk acoustic wave devices, capacitancemeasurements, radioimmunoassay measurements, radiolabeling,chemiluminescence measurements, Lamb-wave measurements, fluorescencemeasurements, Wilhelmy balance, chemiresistor measurements,electrochemical sensors, enzyme-linked immunosorbent assay, resistance,capacitance, acoustic wave, surface plasmon resonance, scanning tunelingmicroscopy, atomic force microscopy, scanning electron microscopy andother techniques known to those of skill in the art for detecting orquantifying macromolecules such as those described in U.S. Patent Nos.5,306,644; 5,313,264; 5,955,729; and 5,976,466.

In one representative embodiment, captured macromolecules can bedetected or quantified by measuring the change in ultraviolet absorbenceof the array of imprints before and after capture. Alternatively, thechange in resistance or capacitance of the array before and aftercapture can be used to detect captured macromolecules or quantify theamount of captured macromolecules. In another embodiment, a plurality ofmacromolecules can be radioactively labeled by covalent modificationwith a radioactive reagent or by synthesizing the macromolecules fromradioactively labeled precursors. Captured macromolecules can then bedetected or quantified by counting radioactive emissions from the arrayby techniques well-known to those of skill in the art.

The relative amounts of a plurality of different macromolecules can bequantified by capturing the plurality of macromolecules and quantifyingthe amount of each macromolecule of the plurality bound to the imprints.In a preferred embodiment, the identity of each imprint is determinablefrom its relative position within the array. An array of imprintswherein the identity of each imprint is determinable can be preparedfrom an array of template molecules wherein the identity of eachtemplate molecule is determinable from its relative position within thearray. Methods of preparing such arrays of template molecules are knownto those of skill in the art, such as those described above.

An array of molecular imprints according to the present invention isuseful for determining the relative amounts of macromolecules from acomplex biological source. This embodiment of the invention specificallyencompasses the evaluation of an expression profile of a cell. In thisembodiment, the complex mixture of macromolecules comprises a pluralityof polypeptides from a cell. An array of imprints is prepared usingtemplate molecules that correspond to portions of the polypeptides ofthe plurality. The plurality of macromolecules is contacted with thearray of imprints. The absolute or relative amount of each macromoleculecaptured by the array of imprints is determined by a method ofquantifying polypeptides known to those of skill in the art. Forexample, the cell that is the source of the plurality of polypeptidescan be grown in the presence of radioactively labeled amino acids. Theamount of each polypeptide bound by the array can then be determined byscintillation counting or by photographic exposure and densitometry.Alternatively, if antibodies are available for the polypeptides of theplurality, the amount of the polypeptides bound by the array of imprintscan be determined by ELISA methods or other methods known to those ofskill in the art. If each imprint of the array is on a discrete matrix,then the amount of each bound polypeptide can be determined directly bya protein assay known to those of skill in the art such as the assaydescribed in Lowry et al., 1951, J. Biol. Chem. 193: 265-220, or theassay described in Bradford, 1976 Anal. Biochem. 72: 248-254. Theexpression profile of the polypeptides of the plurality can be derivedfrom the relative amounts of each polypeptide of the plurality that isbound by the array of imprints.

A macromolecule can be isolated by capturing the macromolecule with amolecular imprint and then recovering the macromolecule from theimprint. The molecular imprint can be an imprint of a template moleculecorresponding to a portion of the macromolecule. Alternatively, if themacromolecule has a structure yet to be determined, the imprint can bean imprint of a template molecule that does not necessarily correspondto a portion of a known macromolecule. The macromolecule can berecovered from the imprint by diffusion or by incubation in urea,guanidine, SDS, or other techniques known to those of skill in the artfor disrupting macromolecular complexes or for denaturingmacromolecules.

5.2.9 Methods of Screening Macromolecules of Unknown or UndeterminedStructure

In another aspect, the present invention is drawn to methods ofscreening macromolecules. This aspect of the invention encompassesscreening both macromolecules of determined structure and screening ofthose of undetermined structure. To screen a plurality ofmacromolecules, the plurality is contacted with an array of imprints, orimprint compositions, as previously described.

At least one imprint of the array is a molecular imprint of a templatemolecule as defined above. If the macromolecules to be screened arepolypeptides, the template molecule should be a peptide or apolysaccharide. If the macromolecules to be screened arepolynucleotides, the template molecule should be a polynucleotide. Ifthe macromolecules to be screened are polysaccharides, the templatemolecule should be a polysaccharide. If the macromolecules to bescreened are a mixture of different classes of macromolecules, the arrayof imprints can comprise imprints of template molecules of thecorresponding classes.

A sample containing a plurality of macromolecules is contacted with thearray of imprints. If any macromolecule of the sample contains astructure that corresponds sufficiently to the structure of the templatemolecule, the macromolecule will be captured by the array of imprints.Any macromolecules captured can be quantified or recovered from theimprint. In instances where the template molecules have structures thatdo not correspond to any portion of the structure of any knownmacromolecule, the present method of screening can be used to capture,isolate, analyze, detect, quantify and/or identify novel macromoleculesfrom complex samples.

6. EXAMPLE 1

Preparation of an Imprint of the C-terminal Sequence of Cytochrome c

In this Example, we describe the preparation of an imprint that willbind the protein cytochrome c. The imprint was prepared using as atemplate molecule the primary sequence of the carboxy-terminus of thepolypeptide chain.

To prepare the imprint, a template molecule was constructed thatcorresponds in sequence to the carboxy-terminal portion of thecytochrome c polypeptide. The last seven amino acids of the horse heartcytochrome c polypeptide (Sigma) have the sequence LKKATNE. AnN-terminal acylated peptide was synthesized with the sequence LKKATNE bystandard techniques. An ethylene glycol dimethylacrylate (EDGMA)solution was prepared by dissolving 2 g EDGMA and 0.4 g acrylamide in 3ml acetonitrile. 20 mg LKKATNE peptide was added to the EDGMA solution.After 20 mg 2,2′-azobisisobutyronitrile (AIBN) was added as a catalyst,the solution was saturated with nitrogen for 5 min and polymerized underultraviolet irradiation at 370 nm for 12 h at 4° C. The resultingimprint polymer was grounded and washed with three changes of 10% aceticacid in methanol for 24 h and then washed with methanol for 3 h and thenthree times with water for 1 h each. The ratio of wash volume to polymervolume was 1:1 for all washes.

7. EXAMPLE 2

Capture of Cytochrome c with an Imprint of its C-terminal Sequence

In this Example we demonstrate that a molecular imprint of a peptidehaving the sequence of the carboxy-terminus of cytochrome c selectivelycaptures the cytochrome c protein from a mixture of proteins.

A protein solution was prepared containing 0.1 mg/ml cytochrome c, 0.1mg/ml trypsin inhibitor, and 0.1 mg/ml carbonic anhydrase (see FIG. 5,lane 1). 0.15 ml of the protein solution was incubated with 0.1 ml ofthe imprint polymer described in Example 1 at room temperatureovernight. 0.15 ml of the protein solution was also incubated with acontrol polymer prepared according to the protocol of Example 1 withoutthe addition of a template molecule.

Both the control polymer and the imprint polymer nonspecifically bound asignificant amount of all of the proteins (see FIG. 5, lanes 3 and 4).Washing with water failed to elute a significant amount of any of thenonspecifically bound proteins (see FIG. 4, lanes 5 and 6). Washing thecontrol polymer with 2% acetic acid removed almost all of the proteins(see FIG. 5, lane 8). In contrast, washing the imprint polymer with 2%acetic acid removed significant amounts of carbonic anhydrase andtrypsin inhibitor only (see FIG. 5, lane 7). Washing with 2% acetic acidremoved trypsin inhibitor and carbonic anhydrase from the imprintpolymer (see FIG. 5, lane 7), but cytochrome c remained tightly bound tothe imprint polymer which retained an orange color. Washing with 2%acetic acid removed all three proteins, including cytochrome c, from thecontrol polymer (see FIG. 5, lane 8).

This Example demonstrates that a imprint of a template moleculeselectively and tightly binds the macromolecule corresponding to thetemplate molecule. Other macromolecules were not bound by the imprint,and the cavities formed by the template molecule associate with themacromolecule to form a complex strong enough to withstand

8. EXAMPLE 3

Preparation of a Conjugate Molecule Comprising a Template MoleculeCorresponding to the Carboxy-terminus of Cytochrome c and a PalmiticAcid Tail Molecule

To create a surface imprint capable of binding the protein cytochrome c,a conjugate molecule corresponding in structure to the sevencarboxy-terminal amino acids of cytochrome c was constructed. A templatemolecule was first designed having the amino acid sequence of the sevencarboxy-terminal amino acids of the horse heart cytochrome cpolypeptide, LKKATNE. A seven amino acid sequence should be sufficientlyunique to provide a surface imprint with specificity for cytochrome c. Apeptide with the sequence LKKATNE was synthesized by standardtechniques.

A conjugate molecule was then prepared with the LKKATNE templatemolecule. Since LKKATNE is a hydrophillic template molecule (see Kyte &Doolittle (1982), J. Mol. Biol. 157:105-132), palmitic acid was chosenas a hydrophobic tail molecule. Palmitic acid was linked to theamino-terminus of the LKKATNE via an amide bond to form apalmitoyl-peptide conjugate molecule.

9. EXAMPLE 4

Preparation of an Acrylamide Surface Imprint Capable of BindingCytochrome c

In this example, we demonstrate the preparation of an acrylamide surfaceimprint capable of binding cytochrome c. The surface imprint is preparedin a two-phase system with the conjugate molecule of Example 3 whosestructure corresponds to the amino acid sequence of the carboxy-terminusof cytochrome c. The conjugate molecule, with a hydrophillic templatemolecule linked to a hydrophobic tail molecule, was designed topartition to the interface of the two-phase system.

Acrylamide monomer solution was prepared by dissolving 28.5 g acrylamideand 1.5 g N-N′-methylene bisacrylamide in 100 ml of 4 M urea. 2 mg ofthe palmitoyl-peptide conjugate molecule of Example 1 was dissolved in 1ml of the acrylamide monomer solution. Ammonium persulfate and TEMEDwere added to the solution as catalysts. The final concentration ofammonium persulfate was 0.02%, and the final concentration of TEMED was0.1%. 0.5 ml light mineral oil was added, and the mixture was sonicatedat 60 watts for 10 min. The resulting suspension was centrifuged at5,000×g for 10 minutes to separate phases. After polymerization at roomtemperature, the mineral oil phase was removed and the polymer waswashed with 10 mM Tris-HCl, pH 9.0, containing 4 M urea and 10% SDS for24 h. The resulting matrix had the form of the interior of an Eppendorftube.

A control polymer was prepared by the same protocol using a controlconjugate molecule prepared with a control template moleculecorresponding to a portion of rabbit skeletal muscle myosin heavy chain.The amino acid sequence of the control template molecule, TKVISEE, isnot found in the primary amino acid sequence of horse heart cytochromec. A palmitic acid tail molecule was linked to the amino terminus of thecontrol template molecule via an amide bond to generate the controlconjugate molecule.

10. EXAMPLE 5

Capture of Cytochrome c with a Polyacrylamide Surface Imprint of itsC-terminal Sequence

In this example we demonstrate that the acrylamide surface imprintprepared in Example 4 with a seven amino acid template moleculeselectively binds the full-length cytochrome c protein.

A 100 μl solution of 0.1 mg/ml bovine serum albumin, 0.1 mg/ml trypsininhibitor, and 0.1 mg/ml cytochrome c (see FIG. 6, lane 1) in MES/ureabuffer (10 mM MES, pH 5.0, and 4 M urea) was incubated withapproximately 0.5 cm² surface imprint of Example 4 at room temperaturefor 4 h. A 100 μl sample of the same protein solution was also incubatedwith a control polymer prepared according to the protocol of Example 4with the control conjugate molecule corresponding to rabbit myosin heavychain (see FIG. 6, lanes 3 and 5). The supernatant was removed (see FIG.6, lanes 2 and 3) and the surface imprint was washed twice with 500 mlMES/urea buffer for 15 min each. Proteins were eluted by washingovernight with 10% SDS in MES/urea buffer (see FIG. 6, lanes 4 and 5).

The supernatant from the surface imprint incubation (see FIG. 6, lane 2)shows significantly more cytochrome c bound the surface imprint comparedto the amount bound by the control polymer (see FIG. 6, 1 ane 3).Washing with MES/urea buffer removed non-specifically bound proteinsfrom the surface imprint and from the control polymer. Elution overnightwith 10% SDS removed a fraction of the cytochrome c specifically boundto the surface imprint (see FIG. 6, lane 4) and some non-specificallybound BSA (see FIG. 6, lanes 4 and 5).

This example demonstrates that the surface imprints of the presentinvention can be used to specifically capture and isolate a protein froma mixture of proteins. This example also demonstrates that the captureof cytochrome c depends on the structure of the template molecule. Thecontrol polymer imprinted with a template molecule that has nocorrespondence to cytochrome c showed no specific binding of cytochromec (see FIG. 6, lane 5).

11. EXAMPLE 6

Preparation of an Acrylamide Surface Imprint Capable of BindingCytochrome c

In this example, we demonstrate the preparation of a second acrylamidesurface imprint capable of binding cytochrome c. The surface imprint isprepared in a two-phase system with the conjugate molecule of Example 3whose structure corresponds to the amino acid sequence of thecarboxy-terminus of cytochrome c. The conjugate molecule, with ahydrophillic template molecule linked to a hydrophobic tail molecule,was designed to partition to the interface of the two-phase system.

Acrylamide monomer solution was prepared by dissolving 28.5 g acrylamideand 1.5 g N-N′-methylene bisacrylamide in 100 ml of 4 M urea. 2 mg ofthe palmitoyl-peptide conjugate molecule of Example 1 was dissolved in 1ml of the acrylamide monomer solution. Ammonium persulfate and TEMEDwere added to the solution as catalysts. The final concentration ofammonium persulfate was 0.02%, and the final concentration of TEMED was0.1%. 0.5 ml light mineral oil was added, and the mixture was sonicatedat 60 watts for 4 min. The resulting suspension was centrifuged at5,000×g for 10 minutes to separate phases. After polymerization at roomtemperature, the mineral oil phase was removed and the polymer waswashed with 10 mM Tris-HCl, pH 9.0, containing 4 M urea and 10% SDS for24 h. The resulting matrix was ground into beads approximately 0.1 mm indiameter.

12. EXAMPLE 7

Capture of Cytochrome c with a Polyacrylamide Surface Imprint of itsC-terminal Sequence

In this example we demonstrate that the acrylamide surface imprintprepared in Example 6 with a seven amino acid template moleculeselectively binds the full-length cytochrome c protein.

A 100 μl solution of 0.1 mg/ml bovine serum albumin, 0.1 mg/ml trypsininhibitor, and 0.1 mg/ml cytochrome c (see FIG. 7, lane 1) in MES/ureabuffer (10 mM MES, pH 5.0, and 4 M urea) was incubated withapproximately 1.5 cm² surface imprint of Example 6 at room temperaturefor 4 h. A 100 μl sample of the same protein solution was also incubatedwith a control polymer prepared according to the protocol of Example 6with no conjugate molecule (see FIG. 7, lanes 1 and 3). The supernatantwas removed (see FIG. 7, lanes 1 and 2) and the surface imprint waswashed twice with 500 ml MES/urea buffer for 15 min each. Proteins wereeluted by washing overnight with 10% SDS in MES/urea buffer (see FIG. 7,lanes 3 and 4).

The supernatant from the surface imprint incubation (see FIG. 7, lane 2)shows significantly more cytochrome c bound the surface imprint comparedto the amount bound by the control polymer (see FIG. 7, lane 1). Washingwith MES/urea buffer removed non-specifically bound proteins from thesurface imprint and from the control polymer. Elution overnight with 10%SDS removed a significant fraction of the cytochrome c specificallybound to the surface imprint (see FIG. 7, lane 4) and somenon-specifically bound BSA (see FIG. 6, lanes 3 and 4).

This example further demonstrates that the surface imprints of thepresent invention can be used to specifically capture and isolate aprotein from a mixture of proteins.

13. EXAMPLE 8

Capture of Cytochrome c from a Cell Lysate with a Polyacrylamide SurfaceImprint of its C-terminal Sequence

In this example we demonstrate that the acrylamide surface imprintprepared in Example 6 with a seven amino acid template moleculeselectively binds the full-length cytochrome c protein from a complexcell lysate.

A 100 μl solution of a cell lysate (1 mg total protein from ratpheochromocytoma cells) spiked with 0.1 mg/ml cytochrome c in MES/ureabuffer (10 mM MES, pH 5.0, and 4 M urea) was incubated withapproximately 1.5 cm² surface imprint of Example 6 at room temperaturefor 4 h. A 100 μl sample of the same protein solution was also incubatedwith a control polymer prepared according to the protocol of Example 6with no conjugate molecule (see FIG. 7, lanes 5 and 7). The supernatantwas removed (see FIG. 7, lanes 5 and 6) and the surface imprint waswashed twice with 500 ml MES/urea buffer for 15 min each. Proteins wereeluted by washing overnight with 10% SDS in MES/urea buffer (see FIG. 7,lanes 7 and 8).

The supernatants from both surface imprint compositions showed that mostproteins of the cell lysate did not bind the compositions (see FIG. 7,lanes 5 and 6). Washing with MES/urea buffer removed non-specificallybound proteins from the surface imprint and from the control polymer.Elution overnight with 10% SDS removed a fraction of the cytochrome cspecifically bound to the surface imprint (see FIG. 7, lane 8). Thecontrol imprint did not specifically bind cytochrome c (see FIG. 7, lane7).

This example demonstrates the powerful specificity of the surfaceimprints of the present invention. The surface imprint of thecarboxy-terminus selectively bound cytochrome c from a complex celllysate. Surface imprints of the present invention can be used to captureand isolate specific macromolecules from the most complex mixtures ofbiological macromolecules.

The present invention is not to be limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention, and any compositions and methods which are functionallyequivalent are within the scope of the invention. Indeed, variousmodifications of the invention in addition to those described above willbecome apparent to those skilled in the art from the foregoingdescription and accompanying drawings. Such modifications are intendedto fall within the scope of the appended claims.

All patents and publications cited herein are hereby incorporated byreference in their entirety.

What is claimed is:
 1. A method of making an imprint composition capableof capturing a macromolecule of interest, comprising the steps of: (a)solidifying a fluidic matrix material capable of retaining shapedcavities when in a solid or semi-solid form in the presence of atemplate molecule that corresponds to a portion of the macromolecule,thereby yielding a solid or semi-solid matrix material having templatemolecules entrapped therein; and (b) removing the template moleculesfrom the solid or semi-solid matrix material, yielding an imprintcomposition capable of capturing the macromolecule.
 2. The method ofclaim 1 in which the fluidic matrix material is a heat-sensitivecomposition and the solidifying step comprises lowering the temperatureof the heat-sensitive composition from above its melting point to belowits melting point.
 3. The method of claim 2 in which the heat-sensitivecomposition is selected from the group consisting of hydrogels, agarose,gelatins and moldable plastics.
 4. The method of claim 1 in which thefluidic matrix material is a composition comprising a polymerizablecompound and the solidifying step comprises polymerizing thepolymerizable compound.
 5. The method of claim 4 wherein thepolymerizable compound is selected from the group consisting of styrene,methyl methacrylate, 2-hydroxyethyl methacrylate, 2-hydroxyethylacrylate, methyl acrylate, acrylamide, vinyl ether, vinyl acetate,divinylbenzene, ethylene glycol dimethacrylate, ethylene glycoldiacrylate, pentaerythritol dimethacrylate, pentaerythritol diacrylate,N,N′-methylenebisacrylamide, N,N′-ethylenebisacrylamide,N,N′-(1,2-dihydroxyethylene)bis-acrylamide and trimethylolpropanetrimethacrylate.
 6. The method of claim 1 in which the template moleculeis immobilized on a solid support or substrate.
 7. The method of claim 1which the solidifying step is carried out under conditions which aredenaturing with respect to the template molecule.
 8. The method of claim1 in which the solidifying step is carried out under conditions whichare native with respect to the template molecule.
 9. The method of claim1, wherein the template molecule corresponds to a terminal portion ofthe macromolecule.
 10. The method of claim 1, wherein the macromoleculeis a polynucleotide and the template molecule comprises anoligonucleotide.
 11. The method of claim 1, wherein the macromolecule isa polypeptide and the template molecule comprises an oligosaccharide ora glycopeptide.
 12. The method of claim 1, wherein the macromolecule isa polypeptide and the template molecule comprises a peptide.
 13. Themethod of claim 12, wherein the sequence of the peptide corresponds to acontiguous sequence of the polypeptide.
 14. The method of claim 13,wherein the peptide is between 3 and 30 amino acids in length.
 15. Themethod of claim 13, wherein the peptide is between 4 and 15 amino acidsin length.
 16. The method of claim 13, wherein the peptide is between 4and 7 amino acids in length.
 17. The method of claim 13, wherein theportion of the polypeptide comprises the C-terminus of the polypeptide,the N-terminus of the polypeptide or an internal sequence of thepolypeptide.